Last Updated 13 Mar 2020


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Spectrophotometry is a vary specific type of Spectroscopy which measures how much light is absorbed by measuring the intensity of the light beam that is not absorbed (transmittance).The word "Spectra" means the range of wavelength, "Photo" means light or photons and "Metry" is the measuring how much light a chemical substance absorbs which it calls the absorbance.

But what we do is measure how much light of the original light beam gets through (transmittance). So, those are related to each other absorbance and percent transmittance mathematically.The basic way to works is the incident light which usually contain different kind of wavelength, for example when we see something have a red color that means the object is absorbs all colors' wavelength except red.

It is helpful to know the color wheel because the color wheel will help you to understand or getting the idea of where in the visible spectrum you would except to see the best absorbance.The work principle of spectrophotometry in (Figure 1): Firstly, we have a light source typically white light contains all wavelengths. We want to collimate the light or make all the wavelengths parallel to one another so, the special collimator or lens can does that, then we pass the light beam through a prism to splits the light into its various wavelength so, for regular white light you get all the colors of the rainbow.

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Spectrophotometer does not just shine all that light at the sample, it shines a vary specific wavelength of light and we can choose that normally by moving a slit in the way of the one wavelength of light or color that we would like to shine through the sample. That particular color of light will then shine through the sample, some of it will be absorbed and some of it will be transmitted. (Io) is the incident light that is the first enters, and (It) is the amount of light that is transmitted through after some has been absorbed.

The remaining light that gets through hits a photocell, photocell is a solid-state detector that picks up how much light, then it prints out on a digital display either absorbance how much was taken away or percent transmittance how much light go through and those two are related. Briefly, you can determine the unknown concentration of the sample by using Beer-Lambert Law which states: there is a linear relationship between the absorbance and the concentration of a sample.

Mathematical formula of Beer's Law is: A=?lcA is the measure of absorbance.? is the molar extinction coefficient or molar absorptivity.l is the path length.-439528256528center842086600left221268Figure 100Figure 1c is the concentration (which is required).There are special techniques for investigating fast reactions which have half-live less than a few secondsLet us take an example for the simplest fast reaction technique (the continuous flow method) which will be used to study the kinetics of the formation of the ferric thiocyanate complex FeSCN+22120900145742100

For the fast reaction between ferric and thiocyanate ions in an acid solution of constant pH, the observed behavior is consistent with the simple mechanism: center2191301Where kf is the bimolecular forward rate constant and kr is the unimolecular reverse rate constant. So, the rate law from this equation is:center27279960Recall that the equilibrium constant K is related to the rate constant by:15775923297435

Where the sign ? means the equilibrium (t=?) value:31439213903453641206384715300At any time (t), Using these relations, and then rewrite the equation in the form:1965852489141700To simplify the integration of this equation, we will choose the experimental conditions such that [Fe+3] ;; [SCN-]. This will allow us to assume that [Fe+3] is essentially constant during the reaction.

The initial conditions are chosen so that [FeSCN+2]0= at t=0 we find:This an approximate solution which becomes exact only when [Fe+3] is constant. In real practice, [Fe+3]0 will be chosen to be ten times larger than [SCN-]0, so that [Fe+3] will be more by about 10 percent during the reaction.2803525690943500-569595690918400If a plot of ln](FeSCN+2)? - (FeSCN+2)[ versus t is linear, then the first order dependence on [SCN-] and [FeSCN+2] is confirmed.

The rate dependence on [Fe+3] has been established as first order. -5779714625Schematic diagram of system for driving reactant solution.00Schematic diagram of system for driving reactant solution.452856889798Spectrophotometry setup00Spectrophotometry setupProcedure for an example of use Spectrophotometer technique in fast reaction: Firstly, turn on the spectrophotometer and leave it warm up before using.

The wavelength setting should be 455 nm throughout the entire experiment. With both reagent stopcocks A and B and the vent stopcock V closed, slowly increase the gas pressure on the reagent solutions until Bourdon pressure gauge indicates about 500 Torr pressures above 1 atm. With the outlet stopcock C open, open and close the reagent stopcocks A and B several times to make sure that both solutions are flowing smoothly and to remove any air bubbles from the system.

Use a beaker to catch the outflow from the capillary tube. Then set the capillary frame at the first fiducial mark which nearest to the mixing chamber, and carry out the three following steps:1- Open Stopcock A and allow the Fe+3 solution to flow for a sufficient time to remove from the capillary tube any solution containing FeSCN+2 species. Then close stopcock A and the outlet stopcock C.2- Open the outlet stopcock C then turn both stopcocks A and B to their fully open positions.

Catch the outflow of solution from the capillary in a beaker until the flow becomes stable. Then quickly switch the outlet tube from the beaker to a volumetric flask and simultaneously start a timer. When It is full, stop the timer and record the time. Return the outlet tube to the beaker. Then carrying out the above flow rate measurement, you should determine the absorbance A of the reaction mixture and record that value together with the distance x from the mixing chamber.

Work quickly to avoid any interference of the reagent solution.3- When both the flow and absorbance measurements are complete, close the outlet stopcock C and then close both stopcock A and B. This is a crucial step in the procedure. If A and B are left open, solution may siphon from one carboy to the other. After a few minutes, determine the absorbance again to obtain the infinite time value. Verify that this value does not change after one more minute.

For the next run, move the capillary support frame so as to line up the second fiducial mark and repeat the first and third steps at this this new distance setting, be careful in moving the capillary support frame.Make two runs at each of the six or seven positions along the capillary tube. Use special care in making the absorbance readings at large values of x.

If time permits, you should also take data at a different driving pressure. Either increase or decrease the gas pressure depending on weather you need more data at low percent reaction or at high, but it may not be safe to exceed about 700 torr overpressures.In this experiment, more of solution A will be used up than solution B if the Fe+3 solution is always used in the first step to make the zero adjustment of the spectrophotometer at each distance setting.

The resulting change in the liquid level for A relative to that for solution B may change the relative flow rates of these solutions. This can be avoided by alternating the use of solution A and B for making the zero adjustments.References:1- Physical chemistry by Gilbert William Castellan.2- Wiley online library. 4- UKessay.5-

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